A fusion proteon of stromal cell-derived factor-1 (sdf-1) and interleukin-2 (il-2) and applications thereof

ABSTRACT

The present invention provides a fusion protein, comprising a chemokine polypeptide, which is a chemokine or a receptor binding domain thereof; and a cytokine polypeptide connected to said chemokine polypeptide, which is an interleukin, a TNF-superfamily cytokine or a receptor-binding domain thereof; wherein the chemokine polypeptide and the cytokine polypeptide have a common target cell, and the fusion protein has an improved chemokine activity as compared to the chemokine polypeptide, and an improved cytokine activity as compared to the cytokine polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No. 16/415,143filed May 17, 2019, which is a Divisional of U.S. application Ser. No.15/658,929 filed on Jul. 25, 2017 (now U.S. Pat. No. 10,336,801), whichis a Continuation of U.S. application Ser. No. 14/649,714, filed on Jun.4, 2015 (abandoned), which is the National Phase under 35 U.S.C. § 371of International Application No. PCT/CN2012/001629, filed on Dec. 5,2012, all of which are hereby expressly incorporated by reference intothe present application.

REFERENCE TO ELECTRONIC SEQUENCE LISTING

The application contains a Sequence Listing which has been submittedelectronically in .XML format and is hereby incorporated by reference inits entirety. Said .XML copy, created on Mar. 6, 2023, is named“5992-0129PUS3.xml” and is 62,561 bytes in size. The sequence listingcontained in this .XML file is part of the specification and is herebyincorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to fusion protein, comprising a chemokineand a cytokine connected thereto, wherein the chemokine and the cytokinehave a mutual target cell, and the fusion protein has an improvedcytokine activity and an improved chemokine activity.

BACKGROUND OF THE INVENTION

Cytokines are a group of proteins that cells release upon excitation(only very few cytokines are expressed on cell membranes). Cytokinesproduced by cells can affect target cells nearby or through bloodcirculation at very low concentration. They have broad functions onpromoting growth, differentiation and activation of target cells. Manycytokines can target immune cells and play a role in immune response.Based on structural and functional differences, cytokines may be broadlydivided into chemokines, interleukins, growth factors, transforminggrowth factors, colony stimulating factors, tumor necrosis factors, andinterferons, etc.

Chemokines are a group of cytokines being able to attract leukocytes,which are generally positively charged, secretory proteins having smallmolecule weights. Their main function is to attract immune cells to aregion having tissue injuries or pathogen infection, allowing leukocytesto subsequently perform phagocytosis or elicit inflammation againstpathogens at this specific site. Leukocytes attracted by chemokines mayinclude neutrophils, monocytes/macrophages, natural killer cells,dendritic cells and other leukocytes, which are of innate immunity; andT lymphocytes (T cells) or B lymphocytes (B cells) of adaptive immunity.Accordingly, chemokines play a very important role in the immune systemof living organisms. Most chemokines have four highly conserved cysteine(C) forming disulfide bonds to stable their structure. Based ondifferent numbers of amino acids between the first two Cs and theprocession of the first C or not, they may be classified into foursubfamilies of CXC (or α), CC (or (β), C (or γ) and CX₃C. Stromalcell-derived factor-1 (SDF-1) is classified into the CXC subfamily ofchemokines, and is also known as CXC ligand 12 (CXCL12). Having beenobserved in many species including humans, mice and cats of mammals andXenopus of amphibians, and zebra fishes, it has little variation betweendifferent species and is highly conserved (Shirozu et al., Genomics 28,495-500). mRNAs transcribed from SDF-1 gene in mice and humans aresubject to different splicings and thus two isoforms of SDF-1 may beobserved: SDF-1α and SDF-1β. The distribution of SDF-1 is very wide, andcan be detected, including in lymphoid tissue, kidney, lung, liver,brain and muscle (Shirozu et al., Genomics 28, 495-500). SDF-1 receptorCXCR4 not only constantly presents in organs, but can also be seen inhematopoietic stem cells, endothelial cells, dendritic cells, B cellsand T cells. Therefore, these cells are attracted by SDF-1 to migrate tothe site with high concentration of the chemokines (Bleul et al.,Nature, 382: 829-833; Oberlin et al., Nature 382: 833-835; Read et al.,Developmental and comparative immunology, 29, 143-152). Interleukin-8(IL-8) is also classified into the CXC subfamily of chemokines (alsoknown as CXCL8). After initial discovery in humans, it was successivelyobserved in economic animals of pigs, cows and chickens. IL-8 at lowconcentration is able to attract several immune cells, includingmonocytes, macrophages, lymphocytes, neutrophils, etc.

CD40 ligand (CD40L) is a member of tumor necrosis factor (TNF)superfamily, which is a cytokine having functions on tumor necrosis andpromoting differentiation, proliferation and apoptosis of white bloodcells. CD40L is synthesized as a transmembrane protein. Take human CD40Las an example, the protein has a total of 261 amino acids, with first 22amino-terminal amino acids being intracellular region, followed by 24amino acids being transmembrane region, and 215 carboxy-terminal aminoacids being extracellular (Exc) region, wherein the Exc region has atits carboxy terminus a TNF homology (TNFh) region conserved for all TNFsuperfamily proteins. CD40L presents mainly in the form of atransmembrane protein on the surface of activated CD4⁺T cells, and alsopresents on CD8⁺T cells, basophils, eosinophils, mast cells, naturalkiller cells, platelets, and even on the surface of CD40-expressingcells.

CD40, receptor of CD40L, is distributed on the surfaces of antigenpresenting cells (APCs) of B cells, dendritic cells, macrophages, etc.Physiologically, these antigen presenting cells can be activated byCD40L expressed by T helper cells, promoting the expression of majorhistocompatibility complex class II (MHC-II) molecules and B7 moleculesto assist in antigen presentation. CD40L activates signal transductionpathways by binding to CD40 on target cells. In addition to theaforementioned promotion of antigen presentation, effecting on B cells,CD40L can promote B cell proliferation, isotype switching ofimmunoglobulins, antibody secretion, memory B cell differentiation, orprevention of apoptosis; effecting on macrophages, CD40L can enhancetheir activation, production of interleukin-12 (IL-12) to activate Thelper 1 (Th1), or secretion of chemokines, or the production of nitricoxide (NO) to promote microorganism defense ability of macrophages;effecting on dendritic cells, it can make them mature and activated,wherein the mature dendritic cells not only express a large amount ofMHC-II molecules to promote antigen presentation, but also secretechemokines of TNF-α and IL-8, macrophage inflammatory protein 1a(MIP-1a), etc.

There are many researches that apply CD40L on vaccine adjuvant ortreatment, for example, as adjuvants for duck hepatitis B virus (DHBV)vaccines (Gares et al., Clin Vaccine Immunol 13, 958-965), humanimmunodeficiency virus (HIV) DNA vaccines (Stone et al., J Virol 80,1762-1772), or in the treatment of human autoimmune diseases (Howard &Miller, Autoimmunity 37, 411-418), etc.

IL-2 is classified into the hematopoietin family, the family including anumber of cell growth-related hormones or other cytokines, etc.Functions of IL-2 include: regulating the maturation and differentiationof T cells, stimulating proliferation and antibody secretion of B cells,promoting cytotoxicity of natural killer cells, and activating monocytesand macrophages, etc. IL-2 can also stimulate T cells and B cells tocontinue expressing MHC, and also stimulate natural killer cells toproduce several different cytokines, including TNF-α, IFN-γ andgranulocyte/macrophage colony stimulating factor (GM-CSF), etc. Studieshave shown that IL-2 has anti-tumor and vaccine-enhancing effects.

However, there remains a need in the art for cytokines and chemokineswith an improved activity.

BRIEF SUMMARY OF THE INVENTION

It was unexpectedly found in the present invention that a fusion proteincomprising a chemokine and a cytokine connected to the chemokine has animproved cytokine activity and an improved chemokine activity.

Accordingly, the present invention provides a fusion protein, comprisinga chemokine polypeptide, which a chemokine or a receptor binding domainthereof; and a cytokine polypeptide connected to the chemokinepolypeptide, which is an interleukin, a TNF-superfamily cytokine, or areceptor binding domain thereof; wherein the chemokine polypeptide andthe cytokine polypeptide have a common target cells, and the fusionprotein has an improved chemokine activity as compared to the chemokinepolypeptide, and an improved cytokine activity as compared to thecytokine polypeptide.

According to the present invention, the chemokine is a CXC chemokine, CCchemokine, C chemokine, and chemokine CX3C, preferably CXC chemokine.According to one embodiment of the present invention, the chemokine maybe a stromal cell derived factor (SDF-1) or IL-8.

According to the present invention, the cytokine polypeptide is aninterleukin, a TNF-superfamily cytokine, or a receptor binding domainthereof In one embodiment of the present invention, the cytokinepolypeptide is IL-2, CD40 ligand, or a receptor binding domain thereof.

In another aspect, the present invention provides an isolated nucleicacid molecule, which encodes a fusion protein of the present invention.

In yet another aspect, the present invention provides an expressionvector, comprising a nucleic acid molecule of the invention.

The present invention also provides a host cell, comprising anexpression vector of the invention or a nucleic acid molecule of theinvention.

Details of various embodiments of the present invention are describedbelow. Other features of the invention will be apparent from thedetailed description of various embodiments and the claims.

Without further elaboration, it is believed that a person of ordinaryskill in the art to which the present invention belongs can utilize theinvention to its broadest extent based on the description above. It isto be understood that the following detailed description are exemplaryand are not restrictive of the other disclosure in any way.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawing. In the drawings:

FIG. 1 is a schematic diagram for chicken CD40L and its derivativeproteins, CD40L_(Exc) and CD40L_(TNFh).

FIG. 2 shows the results of SDS-PAGE and western blot analysis of theexpressed chicken recombinant proteins. Lanel: IL8CD40L_(Exc), expectedsize being 52 kDa; Lane 2: IL8IL2, expected size being 44 kDa; Lane 3:SDF1CD40L_(Exc), expected size being 38 kDa; Lane 4: SDF1CD40LTNFh,expected size being 44 kDa; and Lane 5: SDF1IL2, expected size being 26kDa.

FIG. 3 shows the results of SDS-PAGE and western blot analysis forpurified single proteins. Lane 1: tagged protein, expected size being 21kDa; Lane 2: IL-8, expected size being 13 kDa; Lane 3: SDF-1, expectedsize being 11 kDa; Lane 4: IL-2, expected size being 32 kDa; Lane 5:CD40L_(Exe), expected size being 42 kDa; and Lane 6: CD40L_(TNFh),expected size being 33 kDa.

FIG. 4 shows the chemotaxis of PBMCs by IL-8 derivative proteins. Cellsattracted by the chemokine outside the agar would travel from the centerof the agar to the surrounding. Cells are seen cloudy at lowmagnification. More cells at the surrounding indicates greater degree ofcell chemotaxis. At 2 μM, the chemotaxis extent of IL8CD40L_(Exe) orIL8IL2 was significantly higher than single IL-8 protein.

FIG. 5 shows the chemotaxis of PBMCs by SDF-1 derivative proteins. At 2μM, the chemotaxis extent of SDF1CD40L_(Exc), SDF1CD40L_(TNFh), orSDF1IL2 was significantly higher than single SDF-1 protein.

FIG. 6 shows the activities of CD40L derivative proteins on activatingmacrophages to produce NO. A: CD40L_(Exc); B: CD40L_(TNFh); C:IL8CD40L_(Exc); D: SDF1CD40L_(Exe); E: SDF1CD40L_(TNFh); and the controlgroup: LPS (4 μg/ml) as a positive control group, tagged protein (250nM) and the culture medium as negative control groups. * representsignificantly higher activity as compared to single proteins (*p<0.05,**p<0.01).

FIG. 7 shows the results of test on IL-2 fusion proteins' promotion oflymphocyte proliferation. Stimulation index (SI)=OD of test groups/OD ofthose cultured with RPMI 1640 only. At a concentration of 0.625-160 nM,the SI value of SDF1IL2 fusion protein was significantly higher than theIL-2 alone group (*p<0.05, **p<0.01, ***p<0.001).

FIG. 8 shows that IL-2 fusion protein as an adjuvant of Newcastledisease (ND) vaccine can significantly promote cellular immune response.Chickens of the group administered with IL-2 fusion protein adjuvant andND vaccine exhibited a significantly higher proliferation of memorylymphocytes upon Newcastle disease virus (NDV) antigen re-stimulation,as compared to the ND vaccine group.

FIG. 9 shows that the fusion proteins as adjuvants of infectiousbronchitis (IB) vaccine can significantly promote cellular immuneresponse. Antigen re-stimulation tests were performed for infectiousbronchitis virus (IBV). Chickens of the group administered with IL-2fusion protein adjuvant and IB vaccine exhibited a significantly(p<0.01) higher proliferation of memory lymphocytes, as compared to theIB vaccine group. Chickens of the group administered with CD40L_(Exc)fusion protein adjuvant and IB vaccine exhibited a significantly(p<0.05) higher proliferation of memory lymphocytes, as compared to theIB vaccine group.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person skilled in theart to which this invention belongs.

As used herein, the singular forms “a”, “an”, and “the” refer to one ormore referents unless the context clearly dictates otherwise.

The term “chemokine polypeptide” as used herein refers to a polypeptide,which is a chemokine or a receptor binding domain thereof, wherein thechemokine includes but is not limited to CXC chemokines, CC chemokines,C chemokines and CX₃C chemokines.

The term “cytokine polypeptide” as used herein refers to a polypeptide,which is a cytokine or a receptor binding domain thereof, wherein thecytokine includes but is not limited to interleukins and cytokines ofTNF-superfamily.

The term “chemokine activity” as used herein refers to the activitieswhich chemokines possess or are able to exert in vivo, including but notlimited to, chemotaxis of a variety of immune cells (includingmonocytes, macrophages, T cells , B cells, natural killer cells,dendritic cells and neutrophils, etc.).

The term “cytokine activity” as used herein refers to the activitieswhich cytokines possess or are able to exert in vivo, including but notlimited to the promotion of proliferation, immunoglobulin classswitching and antibody secretion of B cells; differentiation of memory Bcells, or prevention of their apoptosis; promoting macrophages'secretion of interleukin-12 to activate type I helper T cells or secretechemokines; promoting macrophages to produce nitric oxide to enhance thedefense capability against microorganisms; promoting the maturation andactivation of dendritic cells; regulation of the maturation anddifferentiation of T cells; promoting the cytotoxicity and theproduction of a variety of different cytokines of natural killer cells;activation of monocytes and macrophages; and stimulation of T cells andB cells to continuously express MHC, etc.

The present invention provides a fusion protein, comprising a chemokinepolypeptide, which is a chemokine or a receptor binding domain thereof,and a cytokine polypeptide connected to the chemokine polypeptide, whichis a interleukin, a TNF-superfamily cytokine or a receptor bindingdomain thereof; wherein the chemokine polypeptide and the cytokinepolypeptide have a common target cell, and the fusion protein has animproved chemokine activity as compared to the chemokine polypeptide,and an improved cytokine activity as compared to the cytokinepolypeptide.

In preferred embodiments of the present invention, the chemokinepolypeptide and the cytokine polypeptide are connected by a peptidelinker. To join two proteins together and retain their originalconfigurations and functions, an appropriate peptide linker may be addedbetween the two proteins to reduce the interference with each other whenthe proteins fold. And such peptide linker may be a flexible peptidelinker (Gly-Gly-Gly-Gly-Ser)_(n) (usually n is less than 6) with acertain extent of flexibility and hydrophilicity, or a hydrophilichelical peptide linker (Glu-Ala-Ala-Ala-Lys)_(n) (usually n is less than6).

In one embodiment of the invention, the chemokine is a CXC chemokine. Ina certain embodiment, the chemokine is stromal cell-derived factor is(SDF-1). In another certain embodiment, the chemokine is IL-8.

In certain embodiments of the present invention, the chemokinepolypeptide has an amino acid sequence selected from the following: SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, and a homolog thereof and ananalog thereof.

In certain embodiments of the present invention, the cytokine is IL-2,CD40 ligand (CD40L) or a receptor binding domain thereof.

In certain embodiments of the present invention, the cytokinepolypeptide has an amino acid sequence selected from the following: SEQID NO: 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, and a homolog thereof andan analog thereof.

In certain embodiments of the present invention, the fusion protein ofthe present invention has an amino acid sequence selected from thefollowing: SEQ ID NO: 40, 42, 44, 46 and 48.

In another aspect, the present invention provides an isolated nucleicacid molecule, which encodes a fusion protein of the present invention.

In certain embodiments of the present invention, the isolated nucleicacid molecule comprises a nucleotide sequence encoding a chemokinepolypeptide, selected from the following: SEQ ID NO: 1, 3, 5, 7, 9, 11,13, 15, 17, and a homolog thereof and an analog thereof.

In certain embodiments of the invention, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a chemokinepolypeptide, selected from the following: SEQ ID NO: 19, 21, 23, 25, 27,29, 31, 33, 35, 37, and a homolog thereof and an analog thereof.

In certain embodiments of the invention, the isolated nucleic acidmolecule has a sequence of one selected from the following: SEQ ID NO:39, 41, 43, 45 and 47.

In yet another aspect, the present invention provides an expressionvector, comprising a nucleic acid molecule of the invention.

The present invention also provides a host cell comprising an expressionvector of the invention or a nucleic acid molecule of the invention.

The following examples are merely illustrative and not restrictive tothe present invention.

EXAMPLE 1: CONSTRUCTION OF PROKARYOTIC EXPRESSION VECTORS

Previously constructed expression vectors for chicken SDF-1, IL-8, CD40Lderivative proteins and IL-2 (Pei-Shan Wu, National Chung HsingUniversity, Institute of Veterinary Microbiology, 2008 Master's thesis,Studies on chicken CD40L and chemokines; Tsai et al., Taiwan Vet J31:38-45) were used as a template for recombinant polymerase chainreactions, wherein the chicken CD40L (chCD40L) derivative proteinincludes an extracellular domain of CD40L (CD40L_(Exc)) or TNF homologydomain of CD40L (CD40L_(Exc)) (see FIG. 1 ). Genes sequences ofconjugate proteins or fusion proteins SDF1CD40L_(Exc), SDF1CD40L_(TNFh),SDF1IL2, IL8CD40L_(Exc), IL8CD40L_(TNFh), IL8IL2, etc. were expandedtherefrom.

The methods are briefly described as follows. Two specific primer pairswere designed based on the sequences of each of the genes. Forwardprimer of the first pair of primers has a gene sequence of an EcoR Irestriction enzyme site and a front N-terminus of the fusion protein,and the reverse primer has a gene sequence of a helical peptide linkerand a front C-terminus of the fusion protein. This pair of primers canspecifically amplify DNA fragments encoding the front section of thefusion protein and the peptide linker. Forward primer of the second pairof primers has a gene sequence of a helical peptide linker and a rearN-terminus of the fusion protein, and the reverse primer has a genesequence of a Xho I restriction enzyme site and a rear C-terminus of thefusion protein. This pair of primers can specifically amplify DNAfragments encoding the peptide linker and the rear section of the fusionprotein. With the PCR products of this two primer pairs as templates, anadditional PCR was performed using the forward primer of the first pairof primers and the reverse primer of the second pair of primers, andaccordingly the two fragments were connected due to partial overlappingsequences of the helical peptide linker. The products obtained arefusion gene sequence comprising helical peptide linker gene therein.

After treating the above products with EcoR I and Xho I, a ligation withpET vectors (Novagen, Darmstad, Germany) treated by EcoR I and Xho Iusing T4 DNA ligase (Invitrogen) at 16° C. for 16 hour was performed.The constructed prokaryotic expression vector were respectively named aspETSDF1CD40L_(Exc), pETSDF1CD40L_(TNFh), pETSDF1IL2, pETIL8CD40L_(Exc)and pETIL8IL2.

EXAMPLE 2: EXPRESSION OF THE RECOMBINANT PROTEINS

The constructed prokaryotic expression vector was transformed into E.coli expression strain BL21 (DE3), 0.5 mM IPTG was used to induce theexpression of the recombinant protein, and collected bacteria cells bycentrifugation with the removal culture medium. Subsequently, all ofbacteria cells were resuspended in binding buffer, a high pressure celllysis instrument (French Pressure Cell Press, Thermo IEC, Needham,Height, Mass., USA) was used to lysis the bacteria cells, and solubleproteins located in the supernatant after high speed centrifugation wereisolated using nickel ion affinity column.

Insoluble protein located at the bottom pellet after centrifugation weretreated with 8 M to unfold the proteins and they were dissolved in anaqueous solution, which was then subjected to a centrifugation of 12,000rpm 30 minutes and dialysis of the supernatant was performed to slowlydisplace the urea, so that the proteins may refold to their originalconfigurations. Lastly, the proteins were dissolved in phosphate buffercontaining 10% glycerol (H 7.3), filtered through 0.22 μm membrane,concentration was determined by BCA protein assay kit (Pierce, Rockford,Ill., USA), and stored at −20° C. The isolated proteins were identifiedto be correct fusion proteins using MALDI-TOF mass spectrometer.SDS-PAGE and western blot (primary antibody being anti-His antibody,secondary antibody being AP-labeled goat anti-mouse IgG antibody,chromogenic reagent NBT/BCIP) analysis shows that the expressed chickenrecombinant fusion proteins IL8CD40L_(Exc), IL8IL2, SDF1CD40L_(Exc),SDF1CD40L_(TNFh) and SDF1IL2 etc. have the molecular weights asexpected, respectively being 52 kDa, 44 kDa, 38 kDa, 44 kDa and 26 kDa(see FIG. 2 ). In addition, single proteins were also prepared andisolated as controls by the methods as described above (Pei-Shan Wu,National Chung Hsing University, Institute of Veterinary Microbiology,2008 Master's thesis, Studies on chicken CD40L and chemokines; Tsai etal., Taiwan Vet J31: 38-45).

EXAMPLE 3: CHEMOTACTIC ACTIVITY ASSAY

Chemotactic activities of chemokines (SDF-1, or IL-8) and CD40Lderivative proteins or IL-2-fused chemokines were accessed. Peripheralblood mononuclear cells were isolated using Histopaque 1077 (Sigma,Saint Louis, Mo., USA), washed twice with PBS, suspended with 10% FBS inRPMI 1640 (Gibco, Grand Island, N.Y., USA), and then added into 0.6%liquid agar, mixed well, resulting in a final concentration of 0.3% ofthe agar. Subsequently, 2 μl/well of the mixture were dripped in thecenter of the wells on a 48-well plate, and placed in a refrigerator forfive minutes to solidify the agar, thus fixing the cells within theagar. Each well was then added with 250 μl medium containing arespective concentration protein to be tested, cultured overnight beforeobservation.

Based on the minimum effective concentration (MEC) for each protein toexert chemotactic activity, with smaller MEC value indicating betterchemotactic activity, IL-8 fused either with CD40L derivative protein orIL-2 (IL8CD40L_(Exc) or IL8IL2) exhibited a smaller MEC value and abetter chemotactic activity. The fusion proteins have a betterchemotactic activity than IL-8 (see Table 1). SDF-1 fused either withCD40L derivative protein or IL-2 (SDF1CD40L_(Exc) or SDF1IL2) exhibiteda smaller MEC value and a better chemotactic activity. The fusionproteins have a better chemotactic activity than SDF-1 (see Table 1).The chemotactic effects are better in higher concentrations of proteins.At the same concentration, the chemotactic extent of IL8CD40L_(Exc)(with the best chemotactic activity) or IL8IL2 (with the second bestchemotactic activity) were clearly higher than single IL8 protein (seeFIG. 4 ). And at the same concentration, the chemotactic extent ofSDF1CD40LE_(xc), SDF1CD40L_(TNFh) or SDF1IL2 were clearly higher thansimple mixture or single SDF-1 protein (see FIG. 5 ).

TABLE 1 Minimum effective concentrations (MECs) of chemotactic activityGroup Protein MEC A IL-8 125 nM B IL8CD40L_(Exc) 62.5 nM C IL8IL2 62.5nM D SDF-1 125 nM E SDF1CD40L_(Exc) 62.5 nM F SDF1CD40L_(TNFh) 62.5 nM GSDF1IL2 62.5 nM

EXAMPLE 4: ANALYSIS OF ACTIVATION OF MACROPHAGES TO PRODUCE NITRIC OXIDE(NO) BY CD40L DERIVATIVE PROTEINS

Based on CD40L′s property of being able to activate macrophages toproduce NO, CD40L activities of a chemokine fused with a CD40Lderivative protein were assessed. Peripheral blood mononuclear cellswere isolated, washed twice with PBS, suspended in RPMI 1640 containing10% FBS supplemented with 125 ng/ml chicken IL-2 and 4 μg/ml LPS at2×10⁶ cells/ml. One ml of cells were added to each well of a 24-wellplates. One ml of fresh medium (also supplemented with 125 ng/ml chickenIL-2 and 4 μg/ml LPS) were added after 2 days. After 5 day simulationcompleted, monocytes differentiated into macrophages. After PBS washing3 times to remove suspension cells, different concentrations of CD40Lderivative proteins or fusion proteins were added, with the culturemedium, tagged protein expressed by vectors and culture mediumsupplemented with 4 μg/ml LPS as negative and positive control groups.After 48 hour culture, 50 μl culture medium were taken and examined fornitrite (from NO) concentration using a commercially available kit(Griess Reagent System; Promega, Madison, Wis., USA).

IL8CD40L_(Exc) fusion protein exhibited a significantly better activityas compared to the group added CD40L_(Exc) alone (5-30 nM, p<0.05; 180nM, p<0.01). The effects of the fusion protein SDF1CD40L_(Exc) weresignificantly better than CD40L_(Exc) single protein (5 nM, p<0.01;30-180 nM, p<0.05). For the combination of SDF-1 and CD40L_(TNFh),similar results were obtained that SDF1CD40L_(TNFh) fusion protein had asignificantly better effects than a single CD40L_(TNFh) protein (5 nMand 180 nM, p<0.01; 0.8 nM and 30 nM, p<0.05) (see FIG. 6 ).

EXAMPLE 5: ACTIVITY OF IL2 FUSION PROTEIN ON PROMOTING LYMPHOCYTEPROLIFERATION

Based on the activity of IL2 on promoting lymphocyte proliferation, IL-2activities of a chemokine fused with IL-2 were accessed. In view of thatthe activity of intracellular acid phosphatase are proportional to cellnumber, chromogenic substrate p-nitrophenyl phosphate (pNpp) was used.Peripheral blood mononuclear cells were isolated, and then cultured inRPMI 1640 containing 10% FBS supplemented with different concentrationsof proteins, 10 μg/m1 ConA (positive control group), or 10 nM taggedprotein (negative control group) on 96-well plate at 2×10⁵/well. Afterculture for 3 days, the culture was subjected to 3000 rpm centrifugationfor 10 minutes, removed the culture medium, 100 μl chromogenic reagent(0.1 M sodium acetate, H 5.5, 0.1% Triton X-100, and 10 mM pNpp) wereadded to each well, and incubated at 37° C. for two hours. 10 μl 1 NNaOH were then added to terminate the reaction. Absorbance at awavelength of 405 nm was read and used to calculate the stimulationindex (SI), where SI=OD of experimental group/OD of RPMI 1640 cultureonly. The results show that at 0.625-160 nM SDF1IL2 fusion proteinsexhibited significantly higher activity on proliferation promotion thanIL-2 (0.625 nM, p <0.05; 1.25 — 80 nM, p <0.001; 160 nM, p <0.01). Theseresults show that IL-2 activity was significantly improved after fusedwith a chemokine (see FIG. 7 ).

EXAMPLE 6: FUSION PROTEIN AS ADJUVANT OF AVIAN INFECTIOUS BRONCHITIS(IB) VACCINE TO PROMOTE VACCINE-INDUCED IMMUNE RESPONSES

IL-2 fusion protein was used as an adjuvant of avian Newcastle disease(ND) vaccine and administered to chickens. After administration of thevaccine, the blood of the chickens was drawn for the culture oflymphocytes, and inactivated Newcastle disease virus (NDV) were added asantigen to perform antigen re-stimulation assay. 10 μg/ml ConA wereadded to the culture medium as the positive control group. Theproliferation of memory lymphocytes that can recognize NDV antigen ofeach group of chickens was compared. The methods for determiningproliferation state are the same as described in Example 5.Proliferation rate=(OD of test groups/OD of RPMI 1640 cultureonly)×100%. Compared with the group vaccinated with ND vaccine only, thegroups vaccinated with IL-2 fusion proteins as ND vaccine adjuvant(SDF1IL2+ND vaccine) had a significantly enhanced proliferation ofantigen-specific memory lymphocytes upon antigen re-stimulation (seeFIG. 8 ).

EXAMPLE 7: FUSION PROTEIN AS ADJUVANT OF AVIAN INFECTIOUS BRONCHITIS(IB) VACCINE TO PROMOTE VACCINE-INDUCED IMMUNE RESPONSES

IL-2 fusion proteins or CD40L_(Exc) fusion proteins were used as anadjuvant of avian infectious bronchitis (IB) vaccine. Afteradministration of the vaccine, the blood of the chickens was drawn forthe culture of lymphocytes, and inactivated infectious bronchitis virus(IBV) were added as antigen to perform antigen re-stimulation assay. Theproliferation of memory lymphocytes that can recognize IBV antigen ofeach group of chickens was compared. The methods for determiningproliferation state are the same as described in Example 5.Proliferation rate=(OD of test groups/OD of RPMI 1640 cultureonly)×100%. Compared with the group vaccinated with IB vaccine only, thegroups vaccinated with IL-2 fusion proteins as IB vaccine adjuvant(SDF1IL2+IB vaccine) (p<0.01) or vaccinated with CD40L_(Exc) fusionproteins as IB vaccine adjuvant (SDF1CD40L_(Exc)+IB vaccine) (p<0.05)had a significantly enhanced proliferation of antigen-specific memorylymphocytes upon antigen re-stimulation (see FIG. 9 ).

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

1. A method for promoting lymphocyte proliferation in a subject in needthereof, comprising administering to the subject a vaccine compositioncomprising a vaccine, in combination with a fusion protein of a stromalcell-derived factor-1 (SDF-1) and interleukin-2 (IL-2) as an adjuvant,wherein the SDF-1 has the amino acid sequence of SEQ ID NO: 10, 12, 14,16 or 18, and the IL-2 has the amino acid sequence of SEQ ID NO: 30, 32,34, 36 or 38, and the SDF-1 is connected to IL-2 by a hydrophilichelical peptide linker of (Glu-Ala-Ala-Ala-Lys)_(n) where n is less than6, wherein the vaccine composition is administered in an amounteffective in inducing enhancement of lymphocyte proliferation comparedwith the vaccine alone.
 2. The method of claim 1, wherein the vaccine isa Newcastle Disease (ND) vaccine or an Avian Infectious Bronchitis (IB).3. The method of claim 1, wherein the fusion protein has the amino acidsequence of SEQ ID NO:
 44. 4. A method for promoting lymphocyteproliferation in a subject in need thereof, comprising administering tothe subject a vaccine composition comprising a Newcastle Disease (ND)vaccine or an Avian Infectious Bronchitis (IB) vaccine, in combinationwith a fusion protein of a stromal cell-derived factor-1 (SDF-1) andinterleukin-2 (IL-2) as an adjuvant, wherein the fusion protein has theamino acid sequence of SEQ ID NO: 44, and the vaccine composition isadministered in an amount effective in inducing enhancement oflymphocyte proliferation compared with the ND vaccine or the IB vaccinealone.
 5. A method for promoting lymphocyte proliferation, comprisinggrowing lymphocytes in a medium containing a fusion protein of a stromalcell-derived factor-1 (SDF-1) and interleukin-2 (IL-2) wherein the SDF-1has the amino acid sequence of SEQ ID NO: 10, 12, 14, 16 or 18, and theIL-2 has the amino acid sequence of SEQ ID NO: 30, 32, 34, 36 or 38, andthe SDF-1 is connected to IL-2 by a hydrophilic helical peptide linkerof (Glu-Ala-Ala-Ala-Lys)_(n) where n is less than 6, wherein the fusionprotein is present in the medium in an amount effective in inducingimprovement of lymphocyte proliferation compared with the IL-2 alone. 6.The method of claim 4, wherein the fusion protein has the amino acidsequence of SEQ ID NO: 44.